[0001] For many years nasal absorption has been viewed as a potentially attractive route
for administering polypeptides. However, major obstacles limiting sustained interest
in intranasal administration have centered on the difficulty (a) of achieving sufficiently
high levels of absorption and (b) precisely and consistently controlling the amount
of polypeptide received.
[0002] In particular, these difficulties have been apparent in attempting to administer
insulin intranasally. Therefore, it has been accepted generally that treatment of
diabetic patients with insulin can be achieved effectively only by parenteral injection.
Nevertheless, local discomfort and perceived disruption of normal lifestyle deter
many diabetics from readily accepting the present means of insulin treatment. For
these reasons, other routes of insulin administration continue to be sought.
[0003] Over recent years a renewed interest in intranasal administration of insulin has
occurred. In part, this renewed interest is founded upon the development of nebulizers
which consistently deliver a controlled dose of the desired entity to the nasal passage.
Consistent delivery to the nasal mucosa of precisely controlled amounts of insulin
thus can be a reality, and practical intranasal administration of insulin now is largely
dependent upon achieving sufficiently high levels of intranasal insulin absorption.
[0004] The literature to date has suggested that uptake of insulin from the nasal mucosa
is possible only by incorporation into the formulation of additional agsnts. Thus,
Hirai et al., U.S. Patent No. 4,153,689, describe stable aqueous insulin solutions
useful for intranasal administration which have a pH value in the range of from 2.5
to 4.7 and which contain from 0.1 to 20 weight percent of a stabilizing agent selected
from the group consisting. of (a) at least one non-ionic surface active agent whose
hydrophile-lipophile balance value is in the range of from 9 to 22, (b) polyethylene
glycol having a molecular weight in the range of from 200 to 7500 and (c) mixtures
of (a) and (b).
[0005] Hirai, S., Yashiki, T., and Mima, H., Intematl. J. of Pharm. 9,165-172 (1981) describe
enhancement of insulin absorption through the nasal mucosa by addition to the insulin
solution of a surfactant, for example, sodium glycocholate, saponin, or polyoxyethylene-9-lauryl
ether [see, in addition, Hirai, S., Yashiki, T., and Matsuzawa, T., Diabetes 27, 296-299
(1978)].
[0006] Recently, Moses, A.C., Gordon, G.S., Garey. M.C., and Flier, J.S., Diabetes 32, 1040-1047
- (1983), reported data that showed intranasal insulin absorption in humans was achieved
by concomitant administration of bile salt.
[0007] All studies reported to date have focused upon the incorporation in an insulin solution
of an adjuvant, generally an absorption promoter, that is, a surfactant such as a
non-ionic surface active agent a bile salt, or a chelating agent.
[0008] Achieving high levels of insulin absorption m-tranasally now is possible by using
insulin alone when such insulin is in the form of an alkali metal or ammonium salt,
or is a free acid. This invention is directed to this means for treating diabetes
mellitus.
[0009] Furthermore, in the context of the specific insulins used in this invention, further
enhancement of intranasal absorption is possible by incorporating with such insulins,
either alone or jointly, selected absorption enhancing agents.
[0010] In accordance with the invention, them is provided a means for the treatment of diabetes
mellitus which comprises administering to a warm-blooded animal, via the nasal mucous
membrane of said animal having diabetes mellitus a pharmaceutically acceptable amount
of an alkali medal salt, the ammonium salt, or the free acid of a substantially zinc-free
insulin.
[0011] In particular, in one embodiment, them is provided an ivtranasal pharmaceutical formuation
containing insulin for the treatment of diabetes mellitus characterized in that said
intranasal formnlation is comprised of insulin as an alkali metal salt, the ammonium
salt or the free acid of a substntially zinc-free insulin, and a pharmaceutically
acceptable diluent therefor.
[0012] In an additional embodiment this invention is directed to an intranasal pharmaceutical
formulation comprising a pharmaceutically acceptable amount of an alkali metal salt,
the ammonium salt, or the free acid of a substantially zinc-free insulin, and an absorption
enhancing amount of at least one absorption enhancing agent selected from the group
consisting of (1) oleic acid or an ester or salt thereof, (2) a liquid form sorbitan
fatty acid ester, - (3) a liquid form polyoxyethylene derivative of a sorbitan fatty
acid ester, and (4) a liquid form hydroxypolyoxyethylene-polyoxypropyene- polyoxyethylene
copolymer.
Figure 1 is a plot comparing the blood glucose level following intranasal administration
of porcine zinc insulin and porcine sodium insulin at a dose of 4 Units per kilogram
in rats.
Figure 2 is a plot comparing the blood glucose level following intranasal administration
of human zinc insulin and human sodium insulin at a dose of 5 Units per kilogram in
rats.
Figure 3 is a plot comparing the blood glucose level following intranasal administration
of human metal-free insulin and human sodium insulin at a dose of 5 Units per kilogram
in rats.
[0013] As noted, this invention relates to the use of a metal-free insulin or the ammonium
or an alkali metal salt of an insulin. "Alkali metal salt" means, for example, the
sodium salt, the potassium salt, the lithium salt, and the like. Of the foregoing
metal-free insulins and insulin salts, the sodium salt is highly preferred for use
in the method of this invention.
[0014] The insulins used in the method of this invention also are substantially zinc-free.
"Substantially zinc-free" means an insulin to which zinc has not been added.
[0015] The insulins used in the method of this invention can have the structure of any of
a wide range of species, in particular, bovine, porcine, or human, and, most particularly,
human.
[0016] The insulin administered intranasally may be administered as a composition. Compositions
suitable for such intranasal administration conveniently comprise sterile aqueous
solutions or sterile suspensions of the pharmaceutically active ingredient. The sterile
aqueous solution can be isotonic with the blood of the recipient, generally using
sodium chloride, glycerin, glucose, mannitol, sorbitol, and the like. In addition,
the compositions, whether solution or suspension, may contain any of a number of adjuvants,
such as buffers, preservatives, stabilizing agents, agents that promote rapid onset
of action, agents that promote prolonged duration of action, and the like. Typical
preservatives are, for example, phenol, m-cresol, methyl p-hydroxybenzoate, propyl
a-hydroxybenzoate, 1,1,1-trichloro-2-methyl-2-propanol (chlorbutol), benzalkonium
chloride, ethylenediaminetetraacetic acid (EDTA), N,N-dimethyl-N-(2-phenoxyethyl)-l-dodecanaminium
bromide (domiphen bromide), and the like. Although a buffer may be present in the
compositions used, preferably the composition is free of any buffering agent. When
present, however, typical buffers are, for example, sodium phosphate, sodium acetate,
sodium citrate, and the like.
[0017] One or more emulsifying agents may be added to the composition used in the method
of this invention in order to improve its stability. The emulsifier employed may be
chosen from among those generally known to be suitable for use in pharmaceutical formulations.
Typical emulsifiers may be natural emulsifying agents, such as lecithin, and the like.
Synthetic emulsifying agents can also be used. Depending upon the charge possessed
by the emulsifier, the class may be divided into anionic, cationic, and nonionic types.
The preferred emulsifier will be nonionic, and, in particular, a fatty acid, sorbitan
fatty acid ester, or a polyoxyethylene derivative thereof. Examples of such emulsifying
agents are, for example, oleic acid, linoleic acid, linolenic acid, ethyl oleate,
methyl linoleate, methyl linoleate, and the like. A sorbitan fatty acid ester emulsifier
is available commercially under the trademark Span. A polyoxyethylene type of emulsifier
is commercially available under the trademark Tween. A highly preferred emulsifier
is Tween 85, which is polyoxyethylene 20 sorbitan trioleate, and is available from
ICI Americas. Other suitable emulsifiers include the poloxamers including the Pluronic
emulsifiers from BASF Wyandotte such as Pluronic L81. Blends of emulsifiers are common
and may be used also. The reader is referred to REMINGTON PHARMACEUTICAL SCIENCES,
Chapter 21, 16th Edition (1980) for a general discussion of suitable types of emulsifiers
and their uses. If present, the amount of emulsifier employed in compositions used
in the method of this invention generally will be in the range of about 0.005 to about
5.0 percent by weight, more preferably in the range of about 0.05 to about 0.2 percent
by weight of the formulation.
[0018] A dispersion medium also will be present in the intranasal composition used in this
invention. The medium may be composed of a variety of substances such as water and
other similar substantially non-toxic materials. Preferably, however, the medium is
composed of one or more substances also capable of acting as a propellant for the
system. Examples of substances capable of being used are, for example, the Freons
from E.I. DuPont de Nemours and Company such as Freon 114 (1,2-dichloro-1,1,2,2-tetrafluoroethane),
Freon 12 - (dichlorodifluoromethane) and Freon 11 - (trichlorofluoromethane). The
substances preferably are used in combination. While a variety of ingredients in assorted
concentrations may be employed in the combinations, preferred combinations include
about a 1:1 mixture of Freon 11 and Freon 12 by weight and about a 5:4:1 mixture of
Freons 11:12:114 by weight. The amount of ingredients comprising the dispersion medium
will be from about 80.0 to greater than about 99.9 percent by weight, more preferably
from about 95.0 to greater than about 99.9 percent by weight of the total formulation.
[0019] Due to the nasal route of administration, a scenting agent may be added also to the
composition, if desired. When used, the scenting agent will be present in the range
of about 0.001 to about 1.0 percent by weight. Examples of scenting agents are, for
example, peppermint oil, lemon oil, olive oil and other like substances having a soothing
effect on the olfactory system.
[0020] Certain of the previously mentioned emulsifiers unexpectedly are capable of enhancing
intranasal absorption of insulin when present in the insulin formulation. A further
aspect of this invention, therefore, comprises adding to the insulin formulation an
absorption enhancing amount of (1) oleic acid or an ester or salt thereof, (2) a liquid
form sorbitan fatty acid ester, (3) a liquid form polyoxyethylene derivative of a
sorbitan fatty acid ester, or (4) a liquid form hydroxypolyoxyethylene- polyoxypropylene-polyoxyethylene
co-polymer.
[0021] Of the first class of absorption enhancing agents, typical esters are methyl oleate,
ethyl oleate, and the like, and typical salts are alkali metal salts such as sodium
oleate, potassium oleate, and the like. Of the foregoing, oleic acid and ethyl oleate
and preferred, oleic acid being most preferred.
[0022] Typical examples of liquid form sorbitan fatty acid ester absorption enhancing agents
are those available commercially under the trademark Span. Specific examples are Span
20, Span 80, Span 85, and the like. The most preferred such agent is Span 85.
[0023] Typical examples of liquid form polyoxyethylene derivatives of sorbitan fatty acid
ester absorption enhancing agents are those available commercially under the trademark
Tween. Specific examples are Tween 20, Tween 21, Tween 40, Tween 60, Tween 80, Tween
85, and the like. The most preferred such agent is Tween 85.
[0024] Typical examples of liquid form hydroxypolyoxyethylene-polyoxypropylene- polyoxyethylene
co-polymer absorption enhancing agents are those available commercially under the
trademark Pluronic. Specific examples are Pluronic L-31. Pluronic L-35, Pluronic L-42,
Pluronic L-43, Pluronic L-44, Pluronic L-61, Pluronic L-62, Pluronic L-63, Pluronic
L-64, Pluronic L-72, Pluronic L-81, Pluronic L-92, Pluronic L-101, Pluronic L-121,
Pluronic L-122, and the like.
[0025] Of the foregoing classes of absorption enhancing agents, those of the classes represented
by oleic acid and liquid form polyoxyethylene derivatives of sorbitan fatty acid esters
are most preferred.
[0026] When using an absorption enhancing agent from any of these classes, the agent will
be added to the insulin formulation in an amount to achieve enhancement of intranasal
absorption of insulin. Typically, the amount of agent will range, on a weight basis,
from about 025% to about 1% based upon the total final formulation. The preferred
amount generally will be somewhat less and will range from about 025% to about 0.75%
by weight based upon the final formulation.
[0027] Even more preferred formulations will contain an agent from the oleic acid class
in combination with an agent from any of the other three ciasses. The amount of each
agent will be that which is sufficient to achieve enhancement of intranasal absorption
of insulin. The amounts present can be in any of a wide range; typically, however,
each agent will be present in a range on a weight basis of from about 025% to about
1% based upon the final formulation. Preferably, the range will be narrower, each
of the agents being present in the range of about 025% by weight to about 0.75% by
weight. When in this latter range, highly preferred formulations are those having
equivalent weight amounts of each of the agents.
[0028] When the formulations are composed of two absorption enhancing agents, preferably
they are oleic acid (including its esters and salts) and a liquid form polyoxyethylene
derivative of a sorbitan fatty acid ester. Typical highly preferred formulations are
those in which the agents are oleic acid and Tween 85. Most preferably, each agent
witl be present in an amount ranging from about 0.4% by weight to about 0.6% by weight,
and especially being present each in an equivalent amount. A highly useful such formulation
is one containing about 0.5% by weight oleic acid and about 0.5% by weight Tween 85.
[0029] Each of the formulations containing one or more absorption enhancing agents generally
will be formulated such that the final resulting pH is within the range of from about
6 to about 8.
[0030] Formulations of the present invention may be prepared by procedures well known to
those skilled in the art of formulation chemistry. When the formulation contains a
propellant and is in the form of a suspension, it preferably is prepared as follows.
[0031] Prior to formulating, the insulin may be micronized with any of several devices employed
in the art, such as a mechanical micronizer. Next, any ernul- sifying agent, scenting
agent, preservative, and/or other component is combined with the insufm in a suitable
cannister or container. The cannister may be metal or glass, is typical of those employed
in the art for this purpose, and is capable of being pressurized. The cannister then
is charged with one or more propellants as described previously while cooling the
cannister to a temperature in the range of from about 5°C to about -50 °C, and, more
preferably, from about 0°C to about -20°C. The cannister then is sealed, typically
with an aerosol type valve, and warmed to check for leakage. The formulation thus
prepared is then suitable for administration to a warm-blooded animal in need of treatment
for diabetes mellitus.
[0032] Physicians will determine the particular dosage of the insulin to be administered
intranasally. The selected dosages will vary depending upon a number of consideratons
including, for example, the severity of the diabetes mellitus condition, the particular
characteristics of the patient receiving treatment, and the like.
[0033] An aqueous formulation preferably is prepared as follows. A solution containing the
appropriate amount of any selected preservative, buffer, emulsifier, scenting agent,
and/or other component is prepared. A measured amount of insulin is added to the solution,
and sufficient vehicle is added to achieve the desired final concentration.
[0036] The following non-limiting examples illustrate specific intranasal compositions used
in this invention. Also compared is their effectiveness to corresponding zinc insulin
compositions.
[0037] Insulin formulations for Examples 1 to 3 were prepared using the following insulins:
zinc porcine insulin, sodium porcine insulin, zinc human insulin, sodium human insulin,
and metal-free human insulin. The sodium and metal-free insulins were dissolved in
distilled deionized water. For the zinc insulin solutions, 0.05 M KH
2PO. buffer (pH 7.4) or distilled deionized water (adjusted with HCI and NaOH to pH
7.4) was used as solvent. All solutions were prepared to contain about 10 to about
13 Units of insulin per milliliter.
[0038] The formulations were tested in non-fasted male Sprague-Dawley rats (200-300 gm).
On the day of the experiment the rats were transferred to a surgical table where they
could comfortably lie on their back. A surgical procedure was followed as described
in Hussain, A., Hirai, S., and Banarshi, R., J. Pharm. Sci. 69, 1411-1413 (1980);
and Su, K.S.E., Campanale, K.M., and Gries C.L., J. Pharm. Sci. 73, 1251-1254 (1984).
The procedure is summarized as follows. The rats were anesthetized with 50 mg/kg dose
of pentobarbital by intraperitoneal injection 30 minutes prior to surgery. An incision
was made in the neck, and the trachea was cannulated with a polyethylene tube.
[0039] Another tube was inserted from the esophagus to the posterior part of the nasal cavity.
The nasopalatine was closed with an adhesive agent to prevent drainage of the drug
solution from the nasal cavity to the mouth. The drug solution was delivered to the
nasal cavity through the esophagus cannulation tubing by means of a syringe. Twenty
microliters of blood were sampled from the tail vein periodically. The blood sample
was immediately mixed in a sample cup with 480 µl of an anticoagulant solution (prepared
by dissolving 2.0 gm of disodium ethylenediaminetetraacetic acid dihydrate and 0.4
gm of sodium fluoride in deionized water q.s. to 2.0 liters) and assayed by the glucose
oxidase-peroxidase method for the measurement of glucose in serum or plasma. This
method, as modified by P. Trinder Ann. Clin. Biochem., 6, 24 - (1969), and J. Clin.
Pathol., 22, 246 (1969)] involves two reactions to determine glucose concentrations.
First, the oxidation of glucose in the presence of glucose oxidase yields gluconic
acid and peroxide. The peroxide then reacts with phenol and 4-aminoantipyrine in the
presence of peroxidase to yield a red dye measureable at 500-520 nm. The method is
designed for use in automated and semi-automated equipment.
[0040] The protocol for collecting blood samples was designed as follows. A blood sample
was collected from the tail vein at 5, 3, and 1 minute prior to administration of
insulin and at 1, 5, 10, 20, 30, 40, 60, 90, 120, 150 and 180 minutes following administration
of insulin. Each sample was treated as described above.
[0041] The profile of changes in blood glucose level following administration of insulin
was determined as follows. The blood glucose level immediately prior to insulin administration
is represented as 100%. The blood glucose level at time t is expressed as percent
of the pre-injection blood glucose using the equation
[0042] 
in which Ct is blood glucose concentration at time t and Co is blood glucose concentration
immediately prior to insulin administration. For the purpose of relative comparison,
the decrement of the blood glucose level was integrated using the area under the change
in blood glucose versus time curve - (AUC) from 0 to 180 minutes, which corresponds
to the area above the blood glucose concentration curve but under the 100% level.
The AUC was calculated by the trapezoidal method using a computer.
Example 1 -Comparison of Blood Glucose Lowering Effect from Intranasal Administration
of Zinc Porcine Insulin and Sodium Porcine Insulin
[0043] The change in blood glucose levels following intranasal administration of 4 units/kg
of the zinc porcine insulin is shown in Figure 1. The zinc porcine insulin produces
slight hypoglycemic activity. The decline in blood glucose began at about 40 minutes
following intranasal administration and the nadir of blood glucose decline reached
about 16% at 60 minutes and then gradually recovered to the base line. The results
are consistent with those of previous reports which suggest that little or no blood
glucose depression is observed following intranasal administration of zinc insulin
in rats, dogs, and humans. However, when the same dose of sodium porcine insulin (4
unitslkg) was intranasally administered, the blood glucose concentration fell promptly
after 5 to 10 minutes as shown in Figure 1. The nadir of blood glucose occurred at
about 90 minutes, with depression of about 40% or more after which gradual recovery
was observed. Comparison of blood glucose lowering effect after intranasal administration
of zinc porcine insulin and sodium porcine insulin in rats is summarized in Table
1 following. The relative absorption following intranasal administration when compared
to subcutaneous administration is about 23% in the case of zinc porcine insulin and
66% in that of sodium porcine insulin. Moreover, the statistical significance of the
difference of intranasal absorption between zinc porcine insulin and sodium porcine
insulin is p <0.05.

Example 2 --Comparison of Blood Glucose Lowering Effect from Intranasal Administration
of Zinc Human Insulin and Sodium Human Insulin
[0044] The change in blood glucose levels following intranasal administration of 5 units/kg
of zinc human insulin is shown in Figure 2. In a pattern similar to zinc porcine insulin,
the zinc human insulin produced slight blood glucose lowering effect. The decline
in blood glucose began about 20 minutes following intranasal administration. The peak
blood glucose decline reached about 70% of initial at 90 minutes and then gradually
recovered toward the base line; whereas, the peak decline in blood glucose by sodium
insulin was approximately 50%. Comparison of blood glucose lowering effect following
intranasal administration of zinc human insulin and sodium human insulin in rats is
summarized in Table 2. It is apparent that an increased response is available following
intranasal administration of sodium human insulin. Moreover, the statistical significance
of the difference of blood glucose lowering effect between zinc human insulin and
human sodium insulin is p <0.001.

Example 3 -Comparison of Blood Glucose Lowering Effect from Intranasal Administration
of Metal-Free Human Insulin and Sodium Human Insulin
[0045] The change in blood glucose levels following intranasal administration of 5 unitstkg
of metal-free human insulin is shown in Figure 3. In a pattern similar to sodium human
insulin, the metal-free human insulin produced a substantial decline in blood glucose.
Comparison of blood glucose lowering effect following intranasal administration of
sodium human insulin and metal-free human insulin in rats is summarized in Table 3.
It is apparent that the blood glucose lowering response is about the same for both
sodium human insulin and metal-free human insulin administered intranasally.

Example 4 --Intranasal Administration of Sodium Human Insulin Formulations Containing
Absorption Enhancing Agents
[0046] Employing the previously described methods, formulations of sodium human insulin
containing one or more absorption enhancing agents were administered intranasally
to dogs. The formulations were prepared by fitting an appropriate container with a
mechanical stirrer and a cooling bath. The desired amounts of absorption enhancing
agent(s), sodium human insulin, and Freon 11 were mixed in the container until uniform.
An appropriate amount of the resulting insulin suspension concentrate then was weighed
into an aerosol canister. A valve was added and sealed onto the canister. Desired
amounts of Freon 12 and Freon 114 propellants then were added using a pressure-filling
method.
[0047] Three parameters, insulin peak, AUC, and glucose level at nadir, were measured. These
results, in their composite, show the enhancement of absorption by the presence of
one or more of the absorption enhancing agents when compared to formulations lacking
such agents. Table 4 following provides the results from these studies.

1. An intranasal pharmaceutical formulation containing insulin for the treatment of
diabetes mellitus characterized in that said intranasal formulation is comprised of
insulin as an alkali metal salt, the ammonium salt or the free acid of a substantially
zinc-free insulin, and a pharmaceutically acceptable diluent therefor.
2. An intranasal pharmaceutical formulation as claimed in claim 1 characterized further
in that it contains an absorption enhancing amount of at least one absorption enhancing
agent selected from the group consisting of (1) oleic acid or an ester or salt thereof,
(2) a liquid form sorbitan fatty acid ester, (3) a liquid form polyoxyethylene derivative
of a sorbitan fatty acid ester, and (4) a liquid form hydroxypolyoxyethylene-polyoxypropylene-
polyoxyethylene copolymer.
3. A formulation as claimed in claim 2 in which the absorption enhancing agent is
oleic acid or an ester or salt thereof.
4. A formulation as claimed in claim 3 in which oleic acid is present in an amount
of from about .25% to about 1% by weight of the final formulation.
5. A formulation as claimed in claim 2 in which the absorption enhancing agent is
a liquid form polyoxyethylene derivative of a sorbitan fatty acid ester.
6. An intranasal formulation as claimed in claim 5 in which the absorption enhancing
agent is Tween 85 present in an amount of from about .25% to about 1% by weight of
the final formulation.
7. An intranasal formulation as claimed in claim 2 in which the absorption enhancing
agent is present as a combination of (1) oleic acid or an ester or salt thereof and
(2) a liquid form polyoxyethylene derivative of a sorbitan fatty acid ester.
8. An intranasal formulation as claimed in claim 7 in which the absorption enhancing
agent is oleic acid and Tween 85 each present in an amount of from about .25% to about
1 % by weight of the final formulation.
9. An intranasal pharmaceutical formulation as claimed in any one of claims 1 to 8
in which the insulin is sodium insulin.
10. An intranasal pharmaceutical formulation as claimed in claim 9 in which the insulin
is human insulin.
11. An intranasal pharmaceutical formulation which comprises oleic acid and Tween
85, each of which are present in an amount of about .5% by weight based upon the final
formulation, and human sodium insulin.
12. The ammonium salt, an alkali metal salt, or the free acid of a substantially zinc-free
insulin for use in the intranasal treatment of diabetes mellitus.
13. The sodium salt of an insulin for use in the intranasal treatment of diabetes
mellitus.
14. Human insulin as its sodium salt for use in the intranasal treatment of diabetes
mellitus.
15. An intranasal formulation as claimed in any one of claims 1 to 11 for use in the
intranasal treatment of diabetes mellitus.
16. The use of the ammonium salt, an alkali metal salt, or the free acid of a substantially
zinc-free insulin for the manufacture of a medicament for the intranasal treatment
of diabetes mellitus.
17. The use claimed in claim 16 in which the insulin is in the form of its sodium
salt.
18. The use claimed in claim 17 in which the insulin is human insulin.